DNA encoding a human dopamine D1 receptor and uses thereof

ABSTRACT

This invention provides isolated nucleic acid molecules encoding a human dopamine D1 receptor, isolated proteins which are human dopamine D1 receptor, vectors comprising isolated nucleic acid molecules encoding a human dopamine D1 receptor, mammalian cells comprising such vectors, antibodies directed to a human dopamine D1 receptor, nucleic acid probes useful for detecting nucleic acid encoding human dopamine D1 receptor, antisense oligonucleotides complementary to any sequences of a nucleic acid molecule which encodes a human dopamine D1 receptor, pharmaceutical compounds related to human dopamine D1 receptor, and nonhuman transgenic animals which express DNA a normal or a mutant human dopamine D1 receptor. This invention further provides methods for determining ligand binding, detecting expression, drug screening, and treatment involving a human dopamine D1 receptor.

This application is a divisional application of U.S. Ser. No.07/969,267, filed Oct. 5, 1993, now U.S. Pat. No. 5,882,855, which is a371 national stage filing of PCT/US91/04858, filed Jul. 10, 1991, whichis a continuation-in-part of U.S. Ser. No. 07/551,448, filed Jul. 10,1990, now abandoned, the contents of which are hereby incorporated byreference into the present application.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referenced by fullcitations within parentheses. The disclosures of these publications intheir entireties are hereby incorporated by reference in thisapplication in order to more fully describe the state of the art towhich this invention pertains.

Pharmacological studies, and more recently gene cloning, haveestablished that multiple receptor subtypes exist for most, if not all,neurotransmitters. The existence of multiple receptor subtypes providesone mechanism by which a single neurotransmitter can elicit distinctcellular responses. The variation in cellular response can be achievedby the association of individual receptor subtypes with different Gproteins and different signalling systems. Further flexibility isprovided by the ability of distinct receptors for the same ligand toactivate or inhibit the same second messenger system.

Individual receptor subtypes reveal characteristic differences in theirabilities to bind a number of ligands, but the structural basis for thedistinct ligand-binding properties is not known. Physiologists andpharmacologists have attempted to specify particular biologicalfunctions or anatomical locations for some receptor subtypes, but thishas met with limited success. Similarly, the biochemical mechanisms bywhich these receptors transduce signals across the cell surface havebeen difficult to ascertain without having well-defined cell populationswhich express exclusively one receptor subtype.

Dopamine receptors have been classified into two subtypes, D₁ and D₂,based on their differential affinities for dopamine agonists andantagonists, and their stimulation or inhibition of adenylate cyclase(for reviews, see Kebabian, J. W. and Calne, D. B. (1979), Nature 277,93-96; Creese, I., Sibley, D. R., Hamblin, M. W., Leff, S. E. (1983),Ann. Rev. Neurosci. 6, 43-71; Niznik, H. B. and Jarvie, K. R. (1989),Dopamine receptors. in “Receptor Pharmacology and Function”, eds.Williams, M., Glennon, R., and Timmermans, P., Marcel Dekker Inc., NewYork, pp. 717-768). The D₁ receptor of the central nervous system isdefined as an adenylate cyclase stimulatory receptor. The location-ofthe prototypic D₁ receptor is the bovine parathyroid gland, wheredopamine agonists stimulate cAMP synthesis via adenylate cyclase,accompanied by parathyroid hormone release. Dopamine-stimulatedadenylate cyclase activity and parathyroid hormone release are sensitiveto both GTP and cholera toxin. This suggests that the D₁ receptor isassociated with a G_(s) guanine nucleotide binding protein. The D₂receptor, in contrast, inhibits adenylate cyclase activity, and appearsto be the primary target of most neuroleptic drugs (Niznik, H. B. andJarvie, K. R. (1989). Dopamine receptors, in “Receptor Pharmacology andFunction”, eds. Williams, M., Glennon, R., and Timmermans, P., MarcelDekker Inc., New York, pp. 717-768). The prototypic D₂ receptor has beencharacterized in the anterior pituitary where it is associated with theinhibition of release of prolactin and alpha-melanocyte stimulatinghormones. Recent work has shown that several different D₁ and D₂receptor subtypes may be present in the mammalian nervous system(Andersen, P. H., Gingrich, J. A., Bates, M. D., Dearry, A., Falardeau,P., Senogles, S. E., and Caron, M. G. Trends in Pharmacolog. Sci. 11:231 (1990)), which would suggest that a family of different proteinswith pharmacological properties similar to the classically defined D₁and D₂ receptors may exist.

Neuroleptics, in addition to their use as drugs to treat severepsychiatric illnesses, are high affinity ligands for dopamine receptors.Butyrophenones such as haloperidol and spiperone are antagonistsspecific for the D₂ receptor, while the recently developed benzazepinessuch as SCH-23390 and SKF-38393 are selective for the D₁ receptor(Niznik, H. B. and Jarvie, K. R. (1989), Dopamine receptors, in“Receptor Pharmacology and Function”, eds. Williams, M., Glennon, R.,and Timmermans, P., Marcel Dekker Inc., New York, pp. 717-768). Highaffinity D₁ and D₂ selective ligands have conclusively distinguishedthese receptors and made feasible characterization of the receptors inthe central nervous system and peripheral tissues with radioligandbinding techniques. Two types of dopamine receptors, designated D_(A1)and D_(A2), have been identified in the cardiovascular system and aresimilar in their pharmacological characteristics to the brain D₁ and D₂receptors (Niznik, H. B. and Jarvie, K. R. (1989), Dopamine receptors,in “Receptor Pharmacology and Function”, eds. Williams, M., Glennon, R.,and Timmermans, P., Marcel Dekker Inc., New York, pp. 717-768). D_(A1)receptors have been described in renal, mesenteric, splenic, coronary,cerebral, and pulmonary arteries and vascular beds, where dopamineelicits relaxation of vascular smooth muscle. Activation ofcardiovascular D_(A1) receptors appears to stimulate adenylate cyclaseactivity. D_(A2) receptors appear to be localized on preganglionicsympathetic nerve terminals that mediate inhibition of norepinephrinerelease. The molecular relationships among dopamine D₁, D₂, and D_(A2)receptors are unknown.

The need for improved selectivity in the leading D₁ drug class, thebenzazepines (e.g. SKF-38393, SCH-23390 and SCH-23982) recently becameapparent when the strong cross-reactivity of these drugs with theserotonin 5-HT₂ receptor family was uncovered. The 5-HT₂ and 5-HT_(1C)receptors display affinities ranging from 0.2 to 24 nM for SCH-23390 andSCH-23982 (Nicklaus, K. J., McGonigle, P., and Molinoff, P. B. (1988),J. Pharmacol. Exp. Ther. 247, 343-348; Hoyer, D. and Karpf, A. (1988),Eur. J. Pharmacol. 150, 181-184)), raising the possibility thatbehavioral and pharmacological effects ascribed to these drugs may, infact, arise from serotonergic receptor interactions.

The dopamine D₁ receptors belong to a family of receptors which aredistinguished by their seven-transmembrane configuration and theirfunctional linkage to G-proteins. This family includes rhodopsin andrelated opsins (Nathans, J. and Hogness, D. S., Cell 34:807 (1983)), theα and β adrenergic receptors (Dohlman, H. G., et al., Biochemistry26:2657 (1987)), the muscarinic cholinergic receptors (Bonner, T. I., etal., Science 237:527 (1987)), the substance K neuropeptide receptor,(Masu, Y., et al., Nature 329:836 (1987)), the yeast mating factorreceptors, (Burkholder, A. C. and Hartwell, L. H., Nucl. Acids Res.13:8463(1985); Hagan, D. C., et al., Proc. Natl. Acad. Sci. USA 83:1418(1986)); Nakayama, N. et al., EMBO J. 4:2643 (1985)), and the oncogenec-mas, (Young, et al., Cell 45:711 (1986)). Each of these receptors isthought to transduce extracellular signals by interaction with guaninenucleotide-binding (G) proteins (Dohlman, H. G., et al., Biochemistry26:2657 (1987); Dohlman, H. G., et al., Biochemistry 27:1813 (1988);O'Dowd, B. F., et al., Ann. Rev. Neurosci., in press).

The D₂ receptor was recently cloned by Civelli and colleagues (Bunzow,J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J.,Christie, M., Machida, C. A., Neve, K. A., and Civelli, O. (1989),Nature 336: 783-87). This event was soon followed by the discovery of analternatively spliced form (termed D_(2A), D_(2long), D-2_(in), orD₂₍₄₄₄₎) that contains an additional 29 amino acids in the thirdextracellular loop of this receptor (Eidne, K. A. et al. (1989), Nature342: 865; Giros, B. et al. (1989), Nature 342: 923-26; Grandy, D. K. etal. (1989), Proc. Natl. Acad. Sci. USA 86: 9762-66; Monsma, F. J. et al.(1989), Nature 342: 926-29; Chio, C. L. et al. (1990), Nature 343:266-69; Stormann, T. M. et al. (1990), Mol. Pharmacol. 37: 1-6). Asecond dopamine receptor has been cloned which exhibits significanthomology to the D₂ receptor, both in amino acid sequence (75%transmembrane region identity) and in pharmacological properties(Sokoloff, P. et al. (1990), Nature 347: 146-51). This new receptor,termed D₃, is encoded by an intron-containing gene. Unlike the D₂receptor, however, alternatively spliced isoforms of this receptor haveyet to be observed. The D₃ receptor has been shown to serve both as anautoreceptor and as a postsynaptic receptor, and has been localized tolimbic areas of the brain (Sokoloff, P. et al. (1990), Nature 347:146-51). Finally, an intronless gene, quite different in sequence andgene structure from the other two dopamine receptor genes, has beenisolated and identified as a D₁ dopamine receptor subtype (Sunahara, R.K. et al. (1990), Nature 347: 80-83; Zhou, Q.-Y. et al. (1990), Nature347: 76-80; Dearry, A. et al. (1990), Nature 347: 72-76; Monsma, F. J.et al. (1990), Proc. Natl. Acad. Sci. USA 87: 6723-27). This D₁ receptoris predominantly expressed in the rat striatum and olfactory tubercles,and has been shown to couple to stimulation of adenylate cyclaseactivity (Dearry et al. (1990) supra; Monsma et al. (1990) supra;Sunahara et al. (1990) supra; Zhou et al. (1990) supra. Available dataon the G protein-coupled receptor superfamily suggests that the D₁receptor does not exhibit strong sequence homologies to the D₂ receptoror the D₃ receptor. In general, G protein-coupled receptors of the sameneurotransmitter family exhibit closest structural homology to otherfamily members that use the same second messenger pathway. For example,examination of the physiological second messenger pathways of theserotonergic, muscarinic and adrenergic receptors has led severalresearchers to the conclusion that these receptors can be classifiedinto structurally homologous subtypes that parallel their secondmessenger pathways (Bylund, D. B. (1988), Trends Pharmacol. Sci. 9,356-361; Peralta, E. G., Ashkenazi, A., Winslow, J. W., Ramachandran,J., and Capon, D. J. (1988), Nature 334, 434-437; Liao, C.-F., Themmen,A. P. N., Joho, R., Barberis, C., Birnbaumer, M., and Birnbaumer, L.(1989), J. Biol. Chem. 264, 7328-7337; Hartig, P. R. (1989), TrendsPharmacol. Sci. 10, 64-69)). Interestingly, those receptors that coupleto activation of adenylate cyclase appear quite distinct in structurefrom those that inhibit this enzyme activity.

Pharmacological and physiological data have emerged indicating thepresence of further diversity within this receptor family. A D₁ receptorthat stimulates phosphoinositide (PI) hydrolysis in rat striatum hasbeen described (Undie, A. S., and Friedman, E. (1990), J. Pharmacol.Exp. Ther. 253: 987-92) as well as an RNA fraction from the same tissuethat causes dopamine-stimulated PI hydrolysis and intracellular calciumrelease when injected into Xenopus oocytes (Mahan, L. C. et al. (1990),Proc. Natl. Acad. Sci. USA 87: 2196-2200). In addition, two populationsof peripheral D₁ receptor have been described based on differentialsensitivity to sulpiride and several other compounds (Andersen, P. H. etal. (1990), Eur. J. Pharmacol. 137: 291-93). Finally, pharmacologicaldifferences exist within different D₁ receptor tissues that couple toadenylate cyclase-coupled D₁ receptors. Biochemical and pharmacologicaldata suggest further diversity in both the D₁ and D₂ receptorpopulations and indicate that additional dopamine receptor clones remainto be discovered (Andersen et al. (1990) supra).

SUMMARY OF THE INVENTION

This invention provides an isolated nucleic acid molecule encoding ahuman dopamine D₁ receptor.

This invention also provides an isolated protein which is a humandopamine D₁ receptor, an isolated protein having substantially the sameamino acid sequence as the amino acid sequence shown in FIGS. 1A-1E (SEQID NO: 1)

This invention provides a vector comprising an isolated nucleic acidmolecule encoding a human dopamine D₁ receptor.

This invention provides a mammalian cell comprising a DNA moleculeencoding a human dopamine D₁ receptor.

This invention provides a method for determining whether a ligand notknown to be capable of binding to a human dopamine D₁ receptor can bindto a human dopamine D₁ receptor which comprises contacting a mammaliancell comprising a DNA molecule encoding a human dopamine D₁ receptorwith the ligand under conditions permitting binding of ligands known tobind to the dopamine D₁ receptor, detecting the presence of any of theligand bound to the dopamine D₁ receptor, and thereby determiningwhether the ligand binds to the dopamine D₁ receptor.

This invention also provides a method of screening drugs to identifydrugs which specifically interact with, and bind to, the human dopamineD₁ receptor on the surface of a cell which comprises contacting amammalian cell comprising a DNA molecule encoding a human dopamine D₁receptor on the surface of a cell with a plurality of drugs, determiningthose drugs which bind to the mammalian cell, and thereby identifyingdrugs which specifically interact with, and bind to, the human dopamineD₁ receptor.

This invention provides a nucleic acid probe comprising a nucleic acidmolecule of at least 15 nucleotides capable of specifically hybridizingwith a sequence included within the sequence of a nucleic acid moleculeencoding a human dopamine D₁ receptor.

This invention provides an antisense oligonucleotide having a sequencecapable of binding specifically with any sequences of an mRNA moleculewhich encodes a human dopamine D₁ receptor so as to prevent translationof the mRNA molecule.

This invention provides an antibody directed to the human dopamine D₁receptor.

This invention provides a transgenic nonhuman mammal expressing DNAencoding a human dopamine D₁ receptor. This invention also provides atransgenic nonhuman mammal expressing DNA encoding a human dopamine D₁receptor so mutated as to be incapable of normal receptor activity, andnot expressing native dopamine D₁ receptor. This invention furtherprovides a transgenic nonhuman mammal whose genome comprises antisenseDNA complementary to DNA encoding a human dopamine D₁ receptor so placedas to be transcribed into antisense mRNA which is complementary to mRNAencoding a dopamine D₁ receptor and which hybridizes to mRNA encoding adopamine D₁ receptor thereby reducing its translation.

This invention provides a method of determining the physiologicaleffects of expressing varying levels of human dopamine D₁ receptorswhich comprises producing a transgenic nonhuman animal whose levels ofhuman dopamine D₁ receptor expression are varied by use of an induciblepromoter which regulates human dopamine D₁ receptor expression.

This invention also provides a method of determining the physiologicaleffects of expressing varying levels of human dopamine D₁ receptorswhich comprises producing a panel of transgenic nonhuman animals eachexpressing a different amount of human dopamine D₁ receptor.

This invention provides a method for diagnosing in a subject apredisposition to a disorder associated with the expression of aspecific human dopamine D₁ receptor allele which comprises a. isolatingDNA from victims of the disorder, b. digesting the isolated DNA of stepa with at least one restriction enzyme, c. electrophoreticallyseparating the resulting DNA fragments on a sizing gel, d. contactingthe resulting gel with a nucleic acid probe capable of specificallyhybridizing to DNA encoding a human dopamine D₁ receptor and labelledwith a detectable marker, e. detecting labelled bands which havehybridized to the DNA encoding a human dopamine D₁ receptor labelledwith a detectable marker to create a band pattern specific to the DNA ofvictims of the disorder, f. preparing the subject's DNA by steps a-e toproduce detectable labeled bands on a gel, and g. comparing the bandpattern specific to the DNA of victims of the disorder of step e and thesubject's DNA of step f to determine whether the patterns are the sameor different and thereby to diagnose predisposition to the disorder ifthe patterns are the same. This method may also be used to diagnose adisorder associated with the expression of a specific human dopamine D₁receptor allele.

This invention provides a method of preparing the isolated dopamine D₁receptor which comprises inducing cells to express dopamine D₁ receptor,recovering the receptor from the resulting cells, and purifying thereceptor so recovered.

This invention provides a method of preparing the isolated dopamine D₁receptor which comprises inserting nucleic acid encoding dopamine D₁receptor in a suitable vector, inserting the resulting vector in asuitable host cell, recovering the receptor produced by the resultingcell, and purifying the receptor so recovered.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E Nucleotide and deduced amino acid sequence of the geneGL-30. (Also Seq. ID No. 1).

Numbers above the nucleotide sequence indicate nucleotide position. DNAsequence was determined by the chain termination method of Sanger, etal., on denatured doubled-stranded plasmid templates using the enzymeSequenase. Deduced amino acid sequence (single letter code) of a longopen reading frame is shown.

FIGS. 2A-2E Comparison of the Dopamine D₁ (GL-30) receptor primarystructure with other G-protein-coupled receptors. Amino acid sequences(single letter code) are aligned to optimize homology. GL-30 is thehuman dopamine receptor of this invention; GL-39 is the human dopaminepseudogene; and D₁ is the human dopamine D₁ receptor. (Also Seq. ID Nos.2 to 4, respectively). It should be noted that a clone designated D₅ isthe same sequence as that listed as GL-30. (Sunahara, et al. (April1991) Nature, 350:614-619).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the dopamine receptor family is defined as the group ofmammalian proteins that function as receptors for dopamine. A dopaminereceptor subfamily is defined as a subset of proteins belonging to thedopamine receptor family which are encoded by genes which exhibithomology of 65% or higher with each other in their deduced amino acidsequences within presumed transmembrane regions (linearly contiguousstretches of hydrophobic amino acids, bordered by charged or polar aminoacids, that are long enough to form secondary protein structures thatspan a lipid bilayer). Three human dopamine receptor subfamilies can bedistinguished based on the information presently available. The dopamineD₂ receptor subfamily contains the dopamine D₂ receptor. There arecurrently two forms of this receptor which are generated by alternativesplicing mechanisms (Toso, R. D., Sommer, B, Ewert, M, et al. (1989)EMBO 8:4025-4034; Chio, C. L. et al. (1990) Nature 343:266-269; Monsma,F. J. (1990) Nature 342:926-929). The dopamine D₃ receptor whichexhibits significant homology to the D₂ receptor both in amino acidsequence and pharmacological properties (Sokoloff, P. et al. (1990)supra). The human dopamine D₁ receptor subfamily contains the humandopamine D₁ receptor gene GL-30 which is described herein, and the humandopamine D₁ receptor, not yet cloned or isolated, which represents thehuman counterpart of the rat D₁ clone (Sunahara R. K. (1990) Nature347:80-83). Therefore, the term “human dopamine D₁ receptor” as usedherein is defined as meaning a member of the dopamine D₁ receptorsubfamily described above. Although this definition differs from thepharmacological definition used earlier, there is significant overlapbetween the present definition and the pharmacological definition.Members of the human dopamine D₁ receptor subfamily so described includethe dopamine D₁ receptor clone known as GL-30 (which is also known asdopamine D_(1B) receptor subtype) and any other receptors which have a65% or greater transmembrane homology to the DNA and amino acid sequenceshown in FIGS. 1A-1E (SEQ ID NO: 1) according to the definition of“subfamily”. This invention relates to the discovery of the first memberof the human dopamine D₁ receptor subfamily.

This invention provides an isolated nucleic acid molecule such as a DNAmolecule encoding a human dopamine D₁ receptor. Such a receptor is bydefinition a member of the dopamine D₁ receptor subfamily. Therefore,any receptor which meets the defining criteria given above is a humandopamine D₁ receptor. One means of isolating a human dopamine D₁receptor is to probe a human genomic library with a natural orartificially designed DNA probe, using methods well known in the art.DNA probes derived from the human genes encoding dopamine D₁ receptor,for example clone GL-30 is a particularly useful probe for this purpose.DNA and cDNA molecules which encode human dopamine D₁ receptors may beused to obtain complementary genomic DNA, cDNA or RNA from human,mammalian or other animal sources, or to isolate related cDNA or genomicclones by the screening of cDNA or genomic libraries, by methodsdescribed in more detail below. Transcriptional regulatory elements fromthe 5′ untranslated region of the isolated clones, and other stability,processing, transcription, translation, and tissuespecificity-determining regions from the 3′ and 5′ untranslated regionsof the isolated genes are thereby obtained. Examples of a nucleic acidmolecule are an RNA, cDNA, or isolated genomic DNA molecule encoding ahuman dopamine D₁ receptor. Such molecules may have coding sequencessubstantially the same as the coding sequence shown in FIGS. 1A-1E (SEQID NO: 1) or may have coding sequences that are 65% or more homologousto the coding sequence shown in FIG. 1. The DNA molecule of FIGS. 1A-1E(SEQ ID NO: 1) encodes a human dopamine D₁ receptor.

This invention further provides a cDNA molecule encoding a humandopamine D₁ receptor having a coding sequence substantially the same asthe coding sequence shown in FIGS. 1A-1E (SEQ ID NO: 1). This moleculeis obtained by the means described above.

This invention also provides an isolated protein which is a humandopamine D₁ receptor. Examples of such proteins are an isolated proteinhaving substantially the same amino acid sequence as the amino acidsequence shown in FIGS. 1A-1E (SEQ ID NO: 1), which is a human dopamineD₁ receptor. One means for obtaining isolated dopamine D₁ receptor is toexpress DNA encoding the receptor in a suitable host, such as abacterial, yeast, or mammalian cell, using methods well known in theart, and recovering the receptor protein after it has been expressed insuch a host, again using methods well known in the art. The receptor mayalso be isolated from cells which express it, in particular from cellswhich have been transfected with the expression vectors described belowin more detail.

This invention provides a vector comprising an isolated nucleic acidmolecule such as DNA, RNA, or cDNA encoding a human dopamine D₁receptor. Examples of vectors are viruses such as bacteriophages (suchas phage lambda), cosmids, plasmids (such as pUC18, available fromPharmacia, Piscataway, N.J.), and other recombination vectors. Nucleicacid molecules are inserted into vector genomes by methods well known inthe art. For example, insert and vector DNA can both be exposed to arestriction enzyme to create complementary ends on both molecules whichbase pair with each other and are then ligated together with a ligase.Alternatively, linkers can be ligated to the insert DNA which correspondto a restriction site in the vector DNA, which is then digested with therestriction enzyme which cuts at that site. Other means are alsoavailable. An example of a plasmid is a plasmid comprising DNA having acoding sequence substantially the same as the coding sequence shown inFIGS. 1A-1E (SEQ ID NO: 1) and designated clone pdopD1-GL-30, depositedwith the American Type Culture Collection under ATCC Accession No.40839.

This deposit was made pursuant to, and in satisfaction of, the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure with the American Type CultureCollection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852.

This invention also provides vectors comprising a DNA molecule encodinga human dopamine D₁ receptor adapted for expression in a bacterial cell,a yeast cell, or a mammalian cell which additionally comprise theregulatory elements necessary for expression of the DNA in thebacterial, yeast, or mammalian cells so located relative to the DNAencoding a human dopamine D₁ receptor as to permit expression thereof.DNA having coding sequences substantially the same as the codingsequence shown in FIGS. 1A-1E (SEQ ID NO: 1) may usefully be insertedinto the vectors to express human dopamine D₁ receptors. Regulatoryelements required for expression include promoter sequences to bind RNApolymerase and transcription initiation sequences for ribosome binding.For example, a bacterial expression vector includes a promoter such asthe lac promoter, and for transcription initiation, the Shine-Dalgarnosequence and the start codon ATG (Maniatis, et al., Molecular Cloning,Cold Spring Harbor Laboratory, 1982). Similarly, a eucaryotic expressionvector includes a heterologous or homologous promoter for RNA polymeraseII, a downstream polyadenylation signal, the start codon ATG, and atermination codon for detachment of the ribosome. Such vectors may beobtained commercially or assembled from the sequences described bymethods well known in the art, for example the methods described abovefor constructing vectors in general. Expression vectors are useful toproduce cells that express the receptor. Certain uses for such cells aredescribed in more detail below.

This invention further provides a plasmid adapted for expression in abacterial, yeast, or, in particular, a mammalian cell which comprises aDNA molecule encoding a human dopamine D₁ receptor and the regulatoryelements necessary for expression of the DNA in the bacterial, yeast, ormammalian cell so located relative to the DNA encoding a human dopamineD₁ receptor as to permit expression thereof. Some plasmids adapted forexpression in a mammalian cell are pSVL (available from Pharmacia,Piscataway, N.J.) and pcEXV-3 (Miller J. and Germain R. N., J. Exp. Med.164:1478 (1986)). Specific examples of such plasmids are a plasmidadapted for expression in a mammalian cell comprising cDNA having codingsequences substantially the same as the coding sequence shown in FIGS.1A-1E (SEQ ID NO: 1) and the regulatory elements necessary forexpression of the DNA in the mammalian cell. Those skilled in the artwill readily appreciate that numerous plasmids adapted for expression ina mammalian cell which comprise DNA encoding human dopamine D₁ receptorsand the regulatory elements necessary to express such DNA in themammalian cell may be constructed utilizing existing plasmids andadapted as appropriate to contain the regulatory elements necessary toexpress the DNA in the mammalian cell. The plasmids may be constructedby the methods described above for expression vectors and vectors ingeneral, and by other methods well known in the art.

This invention provides a mammalian cell comprising a DNA moleculeencoding a human dopamine D₁ receptor, such as a mammalian cellcomprising a plasmid adapted for expression in a mammalian cell, whichcomprises a DNA molecule encoding a human dopamine D₁ receptor and theregulatory elements necessary for expression of the DNA in the mammaliancell so located relative to the DNA encoding a human dopamine D₁receptor as to permit expression thereof. Numerous mammalian cells maybe used as hosts, including the mouse fibroblast cell NIH3T3, CHO cells,HeLa cells, and Ltk- cells. Expression plasmids such as those describedsupra may be used to transfect mammalian cells by methods well known inthe art such as calcium phosphate precipitation, or DNA encoding thesedopamine D₁ receptors may be otherwise introduced into mammalian cells,e.g., by microinjection, to obtain mammalian cells which comprise DNA,e.g., cDNA or a plasmid, encoding a human dopamine D₁ receptor.

This invention provides a method for determining whether a ligand notknown to be capable of binding to a human dopamine D₁ receptor can bindto a human dopamine D₁ receptor which comprises contacting a mammaliancell comprising a DNA molecule encoding a human dopamine D₁ receptorwith the ligand under conditions permitting binding of ligands known tobind to the dopamine D₁ receptor, detecting the presence of any of theligand bound to the dopamine D₁ receptor, and thereby determiningwhether the ligand binds to the dopamine D₁ receptor. Methods forperforming this technique are well known in the art. The DNA in the cellmay have a coding sequence substantially the same as the coding sequenceshown in FIGS. 1A-1E (SEQ ID NO; 1). Preferably, the mammalian cell isnonneuronal in origin. An example of a nonneuronal mammalian cell is anLtk- cell. (Stable cell lines can be produced by cotransfection of anexpression plasmid such as pSVL or pcEXV, into which the DNA of FIGS.1A-1E (SEQ ID NO: 1) has been subcloned, with a plasmid containing thebacterial gene aminoglycoside phosphotransferase into Ltk- cells(American Type Culture Collection, Rockville, Md., Cell Line CCL 1,3)using the calcium phosphate technique (protocol & kit obtained fromSpecialty Media, Inc. Lavallette, N.J.). Clones expressingaminoglycoside transferase can then be selected by the addition of 1mg/ml G418 (Gibco Laboratories, Grand Island, N.Y.) to the culturemedium. The preferred method for determining whether a ligand is capableof binding to the human dopamine D₁ receptors comprises contacting atransfected nonneuronal mammalian cell (i.e. a cell that does notnaturally express any type of dopamine or G-protein coupled receptor,thus will only express such a receptor if it is transfected into thecell) expressing a dopamine D₁ receptor on its surface, or contacting amembrane preparation derived from such a transfected cell, with theligand under conditions which are known to prevail, and thus to beassociated with, in vivo binding of the ligands to a dopamine D₁receptor, detecting the presence of any of the ligand being tested boundto the dopamine D₁ receptor on the surface of the cell, and therebydetermining whether the ligand binds to the dopamine D₁ receptor.(Methods for so doing are well known in the art, for example a tritiatedligand can be used as a radioligand to detect binding to membranefractions isolated from either transiently or stably transfected celllines which express human dopamine D₁ receptor. Tritiated SCH-23390(71.3 Ci/mMol; Dupont-NEN), which is known in the art to bind with highaffinity to the dopamine D₁ receptor, is used as a radioligand to detectthe expression of the dopamine D₁ gene product in membrane fractionsisolated from either transiently or stably transfected cell lines. Theincubation buffer contains 50 mM Tris-HCl pH 7.4; 120 mM NaCl, 5 mM KCl,1 mM MgCl₂; 2mM CaCl₂; 0.1% ascorbic acid; and 1 μM pargyline, andincubation is initiated by adding cell membranes 10-50 μg/well to a 96well microtiter plate containing tritiated SCH-23390 (finalconcentration 5 nM) in a final volume of 250 μl. After incubating 20minutes at 37° C. in the dark, incubation is terminated by rapidfiltration with a Brandel Model 48R Cell Harvester (Brandel,Gaithersville, Md.). The tritiated SCH-23390 that has bound to thedopamine receptors on the cell membrane is retained on the filters,which are placed in scintillation vials with a scintillation fluid (suchas Ready Safe, Beckman Instruments, Fullerton, Calif.) and counted in ascintillation counter (such as a Beckman LS5000 TA). Specific binding oftritiated SCH-23390 is determined by defining nonspecific binding with10⁻⁶ M cis(-) flupentixol. To determine whether a ligand binds todopamine D₁ receptor, the ligand can be tritiated by methods well knownin the art, and the technique described above for SCH-23390 bindingperformed. But a more efficient method is to perform competitionstudies. The method described above is performed, however in addition totritiated SCH-23390, a different unlabeled ligand is added to each wellof the incubation except control wells. Ligands are initially screenedat a concentration of 1-10 times their reported K_(i) values fordopamine D₁ receptor binding by liquid scintillation spectroscopy in aBeckman LS5000 TA scintillation counter using Ready Safe liquidscintillation cocktail (Beckman Instruments, Fullerton, Calif.) at anefficiency of 50-55% Whichever ligand reduces the counts ofradioactivity over the counts of tritiated SCH-23390 alone hascompetitively reduced binding of the tritiated SCH-23390 by itselfbinding to the dopamine receptor). This response system is obtained bytransfection of isolated DNA into a suitable host cell containing thedesired second messenger system such as phosphoinositide hydrolysis,adenylate cyclase, guanylate cyclase or ion channels. Such a host systemis isolated from pre-existing cell lines, or can be generated byinserting appropriate components of second messenger systems intoexisting cell lines. Such a transfection system provides a completeresponse system for investigation or assay of the activity of humandopamine D₁ receptors with ligands as described above. Transfectionsystems are useful as living cell cultures for competitive bindingassays between known or candidate drugs and ligands which bind to thereceptor and which are labeled by radioactive, spectroscopic or otherreagents. Membrane preparations containing the receptor isolated fromtransfected cells are also useful for these competitive binding assays.Functional assays of second messenger systems or their sequelae intransfection systems act as assays for binding affinity and efficacy inthe activation of receptor function. A transfection system constitutes a“drug discovery system” useful for the identification of natural orsynthetic compounds with potential for drug development that can befurther modified or used directly as therapeutic compounds to activateor inhibit the natural functions of the human dopamine D₁ receptor. Thetransfection system is also useful for determining the affinity andefficacy of known drugs at the human dopamine D₁ receptor sites.

This invention also provides a ligand detected by the method describedsupra.

This invention also provides a method of screening drugs to identifydrugs which specifically interact with, and bind to, the human dopamineD₁ receptor on the surface of a cell which comprises contacting amammalian cell comprising a DNA molecule encoding a human dopamine D₁receptor on the surface of a cell with a plurality of drugs, determiningthose drugs which bind to the mammalian cell, and thereby identifyingdrugs which specifically interact with, and bind to, the human dopamineD₁ receptor. The DNA in the cell may have a coding sequencesubstantially the same as the coding sequence shown in FIGS. 1A-1E (SEQID NO: 1). Preferably, the mammalian cell is nonneuronal in origin, suchas an Ltk- cell. Drug candidates are identified by choosing chemicalcompounds which bind with high affinity to the expressed dopamine D₁receptor protein in transfected cells, using radioligand binding methodswell known in the art and described supra. Drug candidates are alsoscreened for selectivity by identifying compounds which bind with highaffinity to one particular dopamine receptor but do not bind with highaffinity to any other dopamine receptor subtype or to any other knownreceptor site. Because selective, high affinity compounds interactprimarily with the target dopamine D₁ receptor site after administrationto the patient, the chances of producing a drug with unwanted sideeffects are minimized by this approach. This invention provides apharmaceutical composition comprising a drug identified by the methoddescribed above and a pharmaceutically acceptable carrier. Once thecandidate drug has been shown to be adequately bio-available following aparticular route of administration, for example orally or by injection(adequate therapeutic concentrations must be maintained at the site ofaction for an adequate period to gain the desired therapeutic benefit),and has been shown to be non-toxic and therapeutically effective inappropriate disease models, the drug may be administered to patients bythat route of administration determined to make the drug bio-available,in an appropriate solid or solution formulation, to gain the desiredtherapeutic benefit.

This invention provides a nucleic acid probe comprising a nucleic acidmolecule of at least 15 nucleotides capable of specifically hybridizingwith a sequence included within the sequence of a nucleic acid moleculeencoding a human dopamine D₁ receptor, for example with a codingsequence included within the sequence shown in FIGS. 1A-1E (SEQ ID NO:1). Nucleic acid probe technology is well known to those skilled in theart who will readily appreciate that such probes may vary greatly inlength and may be labeled with a detectable label, such as aradioisotope or fluorescent dye, to facilitate detection of the probe.Detection of nucleic acid encoding human dopamine D₁ receptors is usefulas a diagnostic test for any disease process in which levels ofexpression of the corresponding dopamine D₁ receptor is altered. DNAprobe molecules are produced by insertion of a DNA molecule whichencodes human dopamine D₁ receptor or fragments thereof into suitablevectors, such as plasmids or bacteriophages, followed by insertion intosuitable bacterial host cells and replication and harvesting of the DNAprobes, all using methods well known in the art. For example, the DNAmay be extracted from a cell lysate using phenol and ethanol, digestedwith restriction enzymes corresponding to the insertion sites of the DNAinto the vector (discussed above), electrophoresed, and cut out of theresulting gel. Examples of such DNA molecules are shown in FIGS. 1A-1E(SEQ ID NO: 1). The probes are useful for ‘in situ’ hybridization or inorder to locate tissues which express this gene family, or for otherhybridization assays for the presence of these genes or their mRNA invarious biological tissues. In addition, synthesized oligonucleotides(produced by a DNA synthesizer) complementary to the sequence of a DNAmolecule which encodes a human dopamine D₁ receptor of are useful asprobes for these genes, for their associated mRNA, or for the isolationof related genes by homology screening of genomic or cDNA libraries, orby the use of amplification techniques such as the polymerase chainreaction.

This invention also provides a method of detecting expression of adopamine D₁ receptor on the surface of a cell by detecting the presenceof mRNA coding for a dopamine D₁ receptor which comprises obtainingtotal mRNA from the cell using methods well known in the art andcontacting the mRNA so obtained with a nucleic acid probe comprising anucleic acid molecule of at least 15 nucleotides capable of specificallyhybridizing with a sequence included within the sequence of a nucleicacid molecule encoding a human dopamine D₁ receptor under hybridizingconditions, detecting the presence of mRNA hybridized to the probe, andthereby detecting the expression of the dopamine D₁ receptor by thecell. Hybridization of probes to target nucleic acid molecules such asmRNA molecules employs techniques well known in the art. In one possiblemeans of performing this method, nucleic acids are extracted byprecipitation from lysed cells and the mRNA is isolated from the extractusing a column which binds the poly-A tails of the mRNA molecules. ThemRNA is then exposed to radioactively labelled probe on a nitrocellulosemembrane, and the probe hybridizes to and thereby labels complementarymRNA sequences. Binding may be detected by autoradiography orscintillation counting. However, other methods for performing thesesteps are well known to those skilled in the art, and the discussionabove is merely an example.

This invention provides an antisense oligonucleotide having a sequencecapable of binding specifically with any sequences of an mRNA moleculewhich encodes a human dopamine D₁ receptor so as to prevent translationof the mRNA molecule. The antisense oligonucleotide may have a sequencecapable of binding specifically with any sequences of the DNA moleculewhose sequence is shown in FIGS. 1A-1E (SEQ ID NO: 1). A particularexample of an antisense oligonucleotide is an antisense oligonucleotidecomprising chemical analogues of nucleotides.

This invention also provides a pharmaceutical composition comprising anamount of the oligonucleotide described above effective to reduceexpression of a human dopamine D₁ receptor by passing through a cellmembrane and binding specifically with mRNA encoding a human dopamine D₁receptor in the cell so as to prevent its translation and apharmaceutically acceptable hydrophobic carrier capable of passingthrough a cell membrane. The oligonucleotide may be coupled to asubstance which inactivates mRNA, such as a ribozyme. Thepharmaceutically acceptable hydrophobic carrier capable of passingthrough cell membranes may also comprise a structure which binds to areceptor specific for a selected cell type and is thereby taken up bycells of the selected cell type. The structure may be part of a proteinknown to bind a cell-type specific receptor, for example an insulinmolecule, which would target pancreatic cells. DNA molecules havingcoding sequences substantially the same as the coding sequence shown inFIGS. 1A-1E (SEQ ID NO: 1) may be used as the oligonucleotides of thepharmaceutical composition.

This invention also provides a method of treating abnormalities whichare alleviated by reduction of expression of a dopamine D₁ receptorwhich comprises administering to a subject an amount of thepharmaceutical composition described above effective to reduceexpression of the dopamine D₁ receptor by the subject. This inventionfurther provides a method of treating an abnormal condition related todopamine D₁ receptor activity which comprises administering to a subjectan amount of the pharmaceutical composition described above effective toreduce expression of the dopamine D₁ receptor by the subject. Severalexamples of such abnormal conditions are dementia, Parkinson's disease,abnormal cognitive functioning such as schizophrenia, tardivedyskinesia, renal failure, and failure of vascular control, abnormalcircadian rhythms, and abnormal visual activity.

Antisense oligonucleotide drugs inhibit translation of mRNA encodingthese receptors. Synthetic oligonucleotides, or other antisense chemicalstructures are designed to bind to mRNA encoding the dopamine D₁receptor and inhibit translation of mRNA and are useful as drugs toinhibit expression of dopamine D₁ receptor genes in patients. Thisinvention provides a means to therapeutically alter levels of expressionof human dopamine D₁ receptors by the use of a synthetic antisenseoligonucleotide drug (SAOD) which inhibits translation of mRNA encodingthese receptors. Synthetic oligonucleotides, or other antisense chemicalstructures designed to recognize and selectively bind to mRNA, areconstructed to be complementary to portions of the nucleotide sequencesshown in FIGS. 1A-1E (SEQ ID NO: 1) of DNA, RNA or of chemicallymodified, artificial nucleic acids. The SAOD is designed to be stable inthe blood stream for administration to patients by injection, or inlaboratory cell culture conditions, for administration to cells removedfrom the patient. The SAOD is designed to be capable of passing throughcell membranes in order to enter the cytoplasm of the cell by virtue ofphysical and chemical properties of the SAOD which render it capable ofpassing through cell membranes (e.g. by designing small, hydrophobicSAOD chemical structures) or by virtue of specific transport systems inthe cell which recognize and transport the SAOD into the cell. Inaddition, the SAOD can be designed for administration only to certainselected cell populations by targeting the SAOD to be recognized byspecific cellular uptake mechanisms which binds and takes up the SAODonly within certain selected cell populations. For example, the SAOD maybe designed to bind to a receptor found only in a certain cell type, asdiscussed above. The SAOD is also designed to recognize and selectivelybind to the target mRNA sequence, which may correspond to a sequencecontained within the sequences shown in FIGS. 1A-1E (SEQ ID NO: 1), byvirtue of complementary base pairing to the mRNA. Finally, the SAOD isdesigned to inactivate the target mRNA sequence by any of threemechanisms: 1) by binding to the target mRNA and thus inducingdegradation of the mRNA by intrinsic cellular mechanisms such as RNAse Idigestion, 2) by inhibiting translation of the mRNA target byinterfering with the binding of translation-regulating factors or ofribosomes, or 3) by inclusion of other chemical structures, such asribozyme sequences or reactive chemical groups, which either degrade orchemically modify the target mRNA. Synthetic antisense oligonucleotidedrugs have been shown to be capable of the properties described abovewhen directed against mRNA targets (J. S. Cohen, Trends in Pharm. Sci.10, 435 (1989); H. M. Weintraub, Sci. Am. January (1990) p. 40). Inaddition, coupling of ribozymes to antisense oligonucleotides is apromising strategy for inactivating target mRNA (N. Sarver et al.,Science 247, 1222 (1990)). An SAOD serves as an effective therapeuticagent when it is administered to a patient by injection, or when thepatient's target cells are removed, treated with the SAOD in thelaboratory, and replaced in the patient. In this manner, an SAOD servesas a therapy to reduce receptor expression in particular target cells ofa patient, in any clinical condition which may benefit from reducedexpression of dopamine D₁ receptor.

This invention provides an antibody directed to the human dopamine D₁receptor, for example a monoclonal antibody directed to an epitope of ahuman dopamine D₁ receptor present on the surface of a cell and havingan amino acid sequence substantially the same as an amino acid sequencefor a cell surface epitope of the human dopamine D₁ receptor included inthe amino acid sequence shown in FIGS. 1A-1E (SEQ ID NO: 1). Amino acidsequences may be analyzed by methods well known in the art to determinewhether they produce hydrophobic or hydrophilic regions in the proteinswhich they build. In the case of cell membrane proteins, hydrophobicregions are well known to form the part of the protein that is insertedinto the lipid bilayer which forms the cell membrane, while hydrophilicregions are located on the cell surface, in an aqueous environment.Therefore antibodies to the hydrophilic amino acid sequences shown inFIGS. 1A-1E (SEQ ID NO: 1) will bind to a surface epitope of a humandopamine D₁ receptor, as described. Antibodies directed to humandopamine D₁ receptor may be serum-derived or monoclonal and are preparedusing methods well known in the art. For example, monoclonal antibodiesare prepared using hybridoma technology by fusing antibody producing Bcells from immunized animals with myeloma cells and selecting theresulting hybridoma cell line producing the desired antibody. Cells suchas SR3T3 cells or Ltk- cells may be used as immunogens to raise such anantibody. Alternatively, synthetic peptides may be prepared usingcommercially available machines and the amino acid sequences shown inFIGS. 1A-1E (SEQ ID NO: 1). As a still further alternative, DNA, such asa cDNA or a fragment thereof, may be cloned and expressed and theresulting polypeptide recovered and used as an immunogen. Theseantibodies are useful to detect the presence of human dopamine D₁receptors encoded by the isolated DNA, or to inhibit the function of thereceptors in living animals, in humans, or in biological tissues orfluids isolated from animals or humans.

This invention provides a pharmaceutical composition which comprises anamount of an antibody directed to the human dopamine D₁ receptoreffective to block binding of naturally occurring ligands to thedopamine D₁ receptor, and a pharmaceutically acceptable carrier. Amonoclonal antibody directed to an epitope of a human dopamine D₁receptor present on the surface of a cell and having an amino acidsequence substantially the same as an amino acid sequence for a cellsurface epitope of the human dopamine D₁ receptor included in the aminoacid sequence shown in FIGS. 1A-1E (SEQ ID NO: 1) are useful for thispurpose.

This invention also provides a method of treating abnormalities whichare alleviated by reduction of expression of a human dopamine D₁receptor which comprises administering to a subject an amount of thepharmaceutical composition described above effective to block binding ofnaturally occurring ligands to the dopamine D₁ receptor and therebyalleviate abnormalities resulting from overexpression of a humandopamine D₁ receptor. Binding of the antibody to the receptor preventsthe receptor from functioning, thereby neutralizing the effects ofoverexpression. The monoclonal antibodies described above are bothuseful for this purpose. This invention additionally provides a methodof treating an abnormal condition related to an excess of dopamine D₁receptor activity which comprises administering to a subject an amountof the pharmaceutical composition described above effective to blockbinding of naturally occurring ligands to the dopamine D₁ receptor andthereby alleviate the abnormal condition. Several examples of suchabnormal conditions are dementia, Parkinson's disease, abnormalcognitive functioning such as schizophrenia, tardive dyskinesia, renalfailure, and failure of vascular control, abnormal circadian rhythms,and abnormal visual activity.

This invention provides a method of detecting the presence of a humandopamine D₁ receptor on the surface of a cell which comprises contactingthe cell with an antibody directed to the human dopamine D₁ receptor,under conditions permitting binding of the antibody to the receptor,detecting the presence of the antibody bound to the cell, and therebythe presence of the human dopamine D₁ receptor on the surface of thecell. Such a method is useful for determining whether a given cell isdefective in expression of dopamine D₁ receptors on the surface of thecell. Bound antibodies are detected by methods well known in the art,for example by binding fluorescent markers to the antibodies andexamining the cell sample under a fluorescence microscope to detectfluorescence on a cell indicative of antibody binding. The monoclonalantibodies described above are useful for this purpose.

This invention provides a transgenic nonhuman mammal expressing DNAencoding a human dopamine D₁ receptor. This invention also provides atransgenic nonhuman mammal expressing DNA encoding a human dopamine D₁receptor so mutated as to be incapable of normal receptor activity, andnot expressing native dopamine D₁ receptor. This invention also providesa transgenic nonhuman mammal whose genome comprises antisense DNAcomplementary to DNA encoding a human dopamine D₁ receptor so placed asto be transcribed into antisense mRNA which is complementary to mRNAencoding a dopamine D₁ receptor and which hybridizes to mRNA encoding adopamine D₁ receptor thereby reducing its translation. The DNA mayadditionally comprise an inducible promoter or additionally comprisetissue specific regulatory elements, so that expression can be induced,or restricted to specific cell types. Examples of DNA are DNA or cDNAmolecules having a coding sequence substantially the same as the codingsequence shown in FIGS. 1A-1E (SEQ ID NO: 1). An example of a transgenicanimal is a transgenic mouse. Examples of tissue specificity-determiningregions are the metallothionein promotor (Low, M. J., Lechan, R. M.,Hammer, R. E. et al. Science 231:1002-1004 (1986)) and the L7 promotor(Oberdick, J., Smeyne, R. J., Mann, J. R., Jackson, S. and Morgan, J. I.Science 248:223-226 (1990)).

Animal model systems which elucidate the physiological and behavioralroles of human dopamine D₁ receptors are produced by creating transgenicanimals in which the expression of a dopamine D₁ receptor is eitherincreased or decreased, or the amino acid sequence of the expresseddopamine Di receptor protein is altered, by a variety of techniques.Examples of these techniques include: 1) Insertion of normal or mutantversions of DNA encoding a human dopamine D₁ receptor or homologousanimal versions of these genes, by microinjection, retroviral infectionor other means well known to those skilled in the art, into appropriatefertilized embryos in order to produce a transgenic animal (Hogan B. etal. Manipulating the Mouse Embryo, A Laboratory Manual, Cold SpringHarbor Laboratory (1986)); 2) Homologous recombination (Capecchi M. R.Science 244:1288-1292 (1989); Zimmer, A. and Gruss, P. Nature338:150-153 (1989)) of mutant or normal, human or animal versions of thegene with the native gene locus in transgenic animals to alter theregulation of expression or the structure of the dopamine D₁ receptor.The technique of homologous recombination is well known in the art. Thistechnique replaces the native gene with the inserted gene and so isuseful for producing an animal that cannot express native receptor butdoes express, for example, an inserted mutant receptor, which hasreplaced the native receptor in the animal's genome by recombination,resulting in underexpression of the receptor (in more detail, mutuallyhomologous regions of the insert DNA and genomic DNA pair with eachother, resulting in the replacement of the homologous regions of genomicDNA and regions between the homologous regions with the insert).Microinjection adds genes to the genome, but does not remove them, andso is useful for producing an animal which expresses its own and addedreceptors, resulting in overexpression of the receptor. One meansavailable for producing a transgenic animal, with a mouse as an example,is as follows: Female mice are mated, and the resulting fertilized eggsare dissected out of their oviducts. The eggs are stored in anappropriate medium such as M2 medium (Hogan B. et al. Manipulating theMouse Embryo, A Laboratory Manual, Cold Spring Harbor Laboratory(1986)). DNA or cDNA encoding a human dopamine D₁ receptor is purifiedfrom a vector (such as plasmid pdopD1-GL-30 described above) by methodswell known in the art. Inducible promoters may be fused with the codingregion of the DNA to provide an experimental means to regulateexpression of the trans-gene. Alternatively or in addition, tissuespecific regulatory elements may be fused with the coding region topermit tissue-specific expression of the trans-gene. The DNA, in anappropriately buffered solution, is put into a microinjection needle(which may be made from capillary tubing using a pipet puller) and theegg to be injected is put in a depression slide. The needle is insertedinto the pronucleus of the egg, and the DNA solution is injected. Theinjected egg is then transferred into the oviduct of a pseudopregnantmouse (a mouse stimulated by the appropriate hormones to maintainpregnancy but which is not actually pregnant), where it proceeds to theuterus, implants, and develops to term. As noted above, microinjectionis not the only method for inserting DNA into the egg cell, and is usedhere only as an example.

Since the normal action of receptor-specific drugs is to activate or toinhibit the receptor, the transgenic animal model systems describedabove are useful for testing the biological activity of potential drugsdirected against the dopamine D₁ receptor even before such drugs becomeavailable. These animal model systems are useful for predicting orevaluating possible therapeutic applications of drugs which activate orinhibit the dopamine D₁ receptor by inducing or inhibiting expression ofthe native or trans-gene and thus increasing or decreasing expression ofnormal or mutant dopamine D₁ receptors in the living animal. Thus, amodel system is produced in which the biological activity of a potentialdrug directed against the dopamine D₁ receptor can be evaluated beforethe actual development of such a drug. The transgenic animals which overor under produce the dopamine D₁ receptor indicate by theirphysiological state whether over or under production of the dopamine D₁receptor is therapeutically useful. The transgenic model system istherefore useful to evaluate potential drug action. For example, it iswell known in the art that a drug such as an antidepressant acts byblocking neurotransmitter uptake, and thereby increases the amount ofneurotransmitter in the synaptic cleft. The physiological result of thisaction is to stimulate reduced production of receptor by the affectedcells, leading eventually to underexpression. Therefore, an animal whichis engineered to underexpress receptor is useful as a test system toinvestigate whether the action of a drug which results inunderexpression is in fact therapeutic. Again, for example, ifoverexpression is found to lead to abnormalities, then a drug which candown-regulate or act as an antagonist to dopamine D₁ receptor isindicated as worth developing. If a promising therapeutic application isuncovered by these animal model systems, activation or inhibition of thedopamine D₁ receptor can be achieved therapeutically either by producingagonist or antagonist drugs directed against the dopamine D₁ receptor,or indeed by any method which increases or decreases the expression ofthe dopamine D₁ receptor.

This invention provides a method of determining the physiologicaleffects of expressing varying levels of human dopamine D₁ receptorswhich comprises producing a transgenic nonhuman animal whose levels ofhuman dopamine D₁ receptor expression are varied by use of an induciblepromoter which regulates human dopamine D₁ receptor expression. Thisinvention also provides a method of determining the physiologicaleffects of expressing varying levels of human dopamine D₁ receptorswhich comprises producing a panel of transgenic nonhuman animals eachexpressing a different amount of human dopamine D₁ receptor. Suchanimals may be produced by introducing different amounts of DNA encodinga human dopamine D₁ receptor into the oocytes from which the transgenicanimals are developed.

This invention also provides a method for identifying a substancecapable of alleviating abnormalities resulting from overexpression of ahuman dopamine D₁ receptor comprising administering the substance to atransgenic nonhuman mammal expressing at least one artificiallyintroduced DNA molecule encoding a human dopamine D₁ receptor anddetermining whether the substance alleviates the physical and behavioralabnormalities displayed by the transgenic nonhuman mammal as a result ofoverexpression of a human dopamine D₁ receptor. Examples of DNAmolecules are DNA or cDNA molecules having a coding sequencesubstantially the same as the coding sequence shown in FIGS. 1A-1E (SEQID NO: 1).

This invention provides a pharmaceutical composition comprising anamount of the substance described supra effective to alleviate theabnormalities resulting from overexpression of dopamine D₁ receptor anda pharmaceutically acceptable carrier.

This invention further provides a method for treating the abnormalitiesresulting from overexpression of a human dopamine D₁ receptor whichcomprises administering to a subject an amount of the pharmaceuticalcomposition described above effective to alleviate the abnormalitiesresulting from overexpression of a human dopamine D₁ receptor.

This invention provides a method for identifying a substance capable ofalleviating the abnormalities resulting from underexpression of a humandopamine D₁ receptor comprising administering the substance to thetransgenic nonhuman mammal described above which expresses onlynonfunctional human dopamine D₁ receptor and determining whether thesubstance alleviates the physical and behavioral abnormalities displayedby the transgenic nonhuman mammal as a result of underexpression of ahuman dopamine D₁ receptor.

This invention also provides a pharmaceutical composition comprising anamount of a substance effective to alleviate abnormalities resultingfrom underexpression of dopamine D₁ receptor and a pharmaceuticallyacceptable carrier.

This invention further provides a method for treating the abnormalitiesresulting from underexpression of a human dopamine D₁ receptor whichcomprises administering to a subject an amount of the pharmaceuticalcomposition described above effective to alleviate the abnormalitiesresulting from underexpression of a human dopamine D₁ receptor.

This invention provides a method for diagnosing in a subject apredisposition to a disorder associated with the expression of aspecific human dopamine D₁ receptor allele which comprises: a. isolatingDNA from victims of the disorder, b. digesting the isolated DNA of stepa with at least one restriction enzyme, c. electrophoreticallyseparating the resulting DNA fragments on a sizing gel, d. contactingthe resulting gel with a nucleic acid probe capable of specificallyhybridizing to DNA encoding a human dopamine D₁ receptor and labelledwith a detectable marker, e. detecting labelled bands which havehybridized to the DNA encoding a human dopamine D₁ receptor labelledwith a detectable marker to create a band pattern specific to the DNA ofvictims of the disorder, f. preparing the subject's DNA by steps a-e toproduce detectable labeled bands on a gel, and g. comparing the bandpattern specific to the DNA of victims of the disorder of step e and thesubject's DNA of step f to determine whether the patterns are the sameor different and thereby to diagnose predisposition to the disorder ifthe patterns are the same. This method may also be used to diagnose adisorder associated with the expression of a specific human dopamine D₁receptor allele. This method makes use of restriction fragment lengthpolymorphisms in the gene of interest, which may itself encode anabnormal phenotype, or may encode or predispose to an abnormal phenotypein one of its allelic forms, or may encode an abnormal phenotype whenpresent in mutant form. A DNA probe is a useful genetic probe for anallelic abnormality. An allele of a gene will have a specificrestriction fragment pattern when its isolated DNA is digested with asingle restriction enzyme or panel of restriction enzymes, because ofpolymorphisms in the areas of the gene which have nucleotide sequencesthat form sites for restriction enzymes. For example, the gene may havethe sequence AATTC which forms the site for the enzyme EcoRI. Its allelemay have in the same area the sequence AAATC. When the isolated DNAcomprising the gene and its allele are digested with EcoRI by methodswell known in the art, the gene will be cut at the site described andthis cut will create a fragment of a length determined by the locationof the next EcoRI site (assuming this is a single-enzyme digest). Theallele will not be cut at this site, therefore the fragment generated bythe digest will be longer. When the DNA digest is run on an agarose orpolyacrylamide sizing gel and hybridized with the detectably labelledDNA probe for the gene, the detectable band visualized on the gel willcorrespond to the length of the restriction fragments produced. If thefragment is the “long” fragment, then this result indicates that theallele is carried by the DNA digested. If the presence of the allelicform of the gene is associated with a predisposition to a phenotypicabnormality, then the predictive power of such an analysis is important.If the abnormality already exists, then this test is useful fordiagnosis and differential diagnosis. An allele is given only as anexample. This method may be used to detect mutations and polymorphismsof a gene of interest, or the gene itself. Methods for isolating DNA(from a source such as a blood or tissue sample, for example) are wellknown in the art. Methods of visualizing a labeled nucleic acid probehybridized to a gel are also well known in the art. For example, the DNAon a gel is denatured with base, incubated with a radioactively labeledprobe, and a filter (usually nitrocellulose) is placed over the gel,transferring the fragments on the gel to the filter. A piece of film islaid over the filter. The fragments which have hybridized to the probewill expose the film and leave a band marking their positions in thegel.

This invention provides a method of preparing the isolated dopamine D₁receptor which comprises inducing cells to express dopamine D₁ receptor,recovering the receptor from the resulting cells, and purifying thereceptor so recovered. An example of an isolated dopamine D₁ receptor isan isolated protein having substantially the same amino acid sequence asthe amino acid sequence shown in FIG. 1. For example, cells can beinduced to express receptors by exposure to substances such as hormones.The cells can then be homogenized and the receptor isolated from thehomogenate using an affinity column comprising, for example, dopamine,or antibody to the dopamine D₁ receptor, or another substance which isknown to bind to the receptor. The resulting fractions can then bepurified by contacting them with an ion exchange column, and determiningwhich fraction contains receptor activity or binds anti-receptorantibodies. These methods are provided as examples, and do not excludethe use of other methods known in the art for isolating proteins.

This invention provides a method of preparing the isolated dopamine D₁receptor which comprises inserting nucleic acid encoding dopamine D₁receptor in a suitable vector, inserting the resulting vector in asuitable host cell, recovering the receptor produced by the resultingcell, and purifying the receptor so recovered. An example of an isolateddopamine D₁ receptor is an isolated protein having substantially thesame amino acid sequence as the amino acid sequence shown in FIGS. 1A-1E(SEQ ID NO: 1). This method for preparing dopamine D₁ receptor usesrecombinant DNA technology methods well known in the art. For example,isolated nucleic acid encoding dopamine D₁ receptor is inserted in asuitable vector, such as an expression vector. A suitable host cell,such as a bacterial cell, or a eucaryotic cell such as a yeast cell, istransfected with the vector. The dopamine D₁ receptor is isolated fromthe culture medium by affinity purification or by chromatography or byother methods well known in the art.

Applicants have identified individual receptor subtype proteins and havedescribed methods for the identification of pharmacological compoundsfor therapeutic treatments. Pharmacological compounds which are directedagainst specific receptor subtypes provide effective new therapies withminimal side effects.

Disturbances of dopaminergic neurotransmission have been associated witha wide range of neurological, endocrine, and psychiatric disorders,including Parkinson's disease, tardive dyskinesia, and schizophrenia.The neuroleptics, which have highest affinity for D₂ receptors havemajor side effects involving movement disorders and hypersecretion ofprolactin. Drugs used in the treatment of Parkinson's disease causenausea, vomiting, choreiform movements, psychiatric disturbancesincluding hallucinations, and cardiovascular disorders. Some of theseeffects are likely to be due to actions on D₁ receptors or to adisruption in the balance of activity between the D₁ and D₂ systems(Abbott, A., 1990; TIPS 11: 49-51). In fact some therapeutic benefit ofD₁ antagonists which lack D₂ activity may be obtained. (Hess, E. J. andCreese, I., in Neurobiology of Central D₁ Receptors, eds, G. R. Breeseand I. Creese pp. 53-72) Drugs selectively targeted to D₁ receptor maybe useful neuroleptics without resulting in the tardive dyskinesiathought to be the result of D₂ receptor up-regulation caused by chronicD₂ antagonism (Hess, E. J. and Creese, I., in Neurobiology of Central D₁Receptors, eds, G. R. Breese and I. Creese pp. 53-72). Furthermore,evidence provided by the anatomical distribution of D₁ receptors in thebrain suggest roles for D₁ selective drugs in cognitive function,control of visual activity and circadian rhythms (Dawson, T., Gelhert,D., McCabe, R., Barnett, A. , and Wamsley, J. 1986; J. Neurosci.6:2352-2365). Finally, the distribution of D₁ receptors on the renalvasculature indicates potential therapeutic value of selective D₁ agentsto ameliorate renal failure secondary to heart attack (Missale, C.,Castelleti, L., Memo, M., Carruba, M., and Spano, P. 1988; J.Cardiovascular Research 11: 643-650.) Its general action on vascularsmooth muscle in other portions of the vascular tree may indicate ageneral role in cardiovascular control (Missale, as above; Hilditch, A.and Drew, G. M. 1985, TIPS 6:396-400).

In animal models, D₁-selective benzazepines induce intense grooming(Molloy, A. G. and Waddington, J. L. (1987), Psychopharmacology (Berlin)92, 164-168), inhibit spontaneous locomotion (Hjorth, S. and Carlsson,A. (1988), J. Neural Transm. 72, 83-97), and generally seem tofacilitate D2 receptor activities (Waddington, J. L. (1986), Biochem.Pharmacol. 35, 3661-3667; Hjorth, S. and Carlsson, A. (1988), J. NeuralTransm. 72, 83-97). These actions produce therapeutic applications inenhancing Parkinson's disease or antipsychotic therapies with existingD₂ antagonists (Waddington, J. L. (1986), Biochem. Pharmacol. 35,3661-3667). In human peripheral arteries, D_(A1) receptors mediatevasodilation (Toda, N., Okunishi, H., and Okamura, T. (1989), Arch Int.Pharmacodyn. Ther. 297, 86-97). Clinical trials with the selectiveD_(A1) receptor agonist, fenoldopam, have revealed a potent renalvasodilatory action that could provide an attractive alternative therapyfor treating hypertensive and congestive heart failure patients (Carey,R. A. and Jacob, L. (1989), J. Clin Pharmacol. 29, 207-211). Developmentof more selective D₁ agonists and antagonists will expand existing D₁therapeutic applications and suggest new ones.

This invention identifies for the first time a new human receptorprotein, the dopamine D₁ receptor, its amino acid sequences, and itshuman gene, clone GL-30. The first isolated human cDNA and genomic cloneencoding dopamine D₁ receptor are identified and characterized herein.The information and experimental tools provided by this discovery willbe useful to generate new therapeutic agents, and new therapeutic ordiagnostic assays for the new receptor protein, associated mRNAmolecules, or associated genomic DNA.

The invention will be better understood by reference to the ExperimentalDetails which follow, but those skilled in the art will readilyappreciate that the specific experiments detailed are only illustrativeof the invention as described more fully in the claims which followthereafter.

Experimental Details

Homology Cloning.

A human spleen genomic library, provided by Dr. Jeffrey V. Ravetch(Sloan-Kettering Institute, New York, N.Y.), was screened using the1.6-kilobase (kb) Xbal-BamHI fragment from the human 5-hydroxytryptamine(⁵-HT_(1A)) receptor gene as a probe. The probe was labeled with ³²p bythe method of random priming. Hybridization was performed at 40° C. in asolution containing 25% formamide, 10% dextran sulfate, 5×SSC (1×SSC is0.15 M sodium chloride, 0.015 M sodium citrate) 1×Denhardt's (0.02%polyvinyl-pyrrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin),and 200 μg/ml of sonicated salmon sperm DNA. The filters were washed at40° C. in 0.1×SSC containing 0.1% sodium-dodecyl-sulfate (SDS) andexposed at −70° C. to Kodak XAR film in the presence of an intensifyingscreen. Lambda phage hybridizing to the probe were plaque purified andDNA was prepared for Southern blot analysis (Maniatis et al., MolecularCloning, Cold Spring Harbor, 1982; E. Southern, J. Mol. Biol. 98:503,1975). For subcloning and further southern blot analysis DNA wasinserted into pUC18 (Pharmacia, Piscataway, N.J.).

DNA Sequencing

Nucleotide sequence analysis was done by the Sanger dideoxy nucleotidechain-termination method (S. Sanger, et al., Proc. Natl. Acad. Sci., 74:5463-5467, 1977) on denatured double-stranded plasmid templates (Chenand Seeburg, DNA 4: 165, 1985) using Sequenase (U.S. Biochemical Corp.,Cleveland, Ohio).

Receptor Expression in Transfected Mammalian Cells

To confirm the functional identity of the newly isolated gene cloneGL-30 was expressed in cultured cell lines. The entire coding region ofGL-30, including 113 base pairs of 5′ untranslated sequence, andapproximately 1.3 kb of 3′ untranslated sequence, was cloned into theeucaryotic expression vector pcEXV-3 (Miller, J. and Germain, R. N.(1986), J. Exp. Med. 164: 1478-89). The resulting plasmid wastransiently transfected into Cos-7 cells using the DEAE-dextranprocedure (Cullen, Methods in Enz., 152: 684-704, 1987).

Measurement of cAMP Formation

The transiently transfected plates were incubated in Dulbecco's modifiedEagle's medium (DMEM, Specialty Media, Lavallette, N.J.), 5mMtheophylline, 10 mM Hepes, 10 μM pargyline, 10 μM propanolol, and/or 10μM SCH-23390 for 20 minutes at 37° C., 5% CO₂. In these experiments, theβ-adrenergic antagonist propanolol was included in the assay to precludestimulation of the endogenous Cos-7 cell β-adrenergic receptor bydopamine. Dopamine or SXF-38393 was then added to a final concentrationof 1 μM and incubated for an additional 10 minutes at 37° C., 5% CO₂.The media was aspirated and the reaction stopped by the addition of 100mM HCl. The plates were stored at 4° C. for 15 minutes, centrifuged for5 minutes, 500×g to pellet cellular debris, and the supernatantaliquotted and stored at −20° C. prior to assessment of cAMP formationby radioimmunoassay (cAMP Radioimmunoassay Kit, Advanced Magnetics,Cambridge, Mass.).

Membrane Preparation

Membranes were harvested from transfected Cos-7 cells which were grownto 100% confluency. The cells were washed twice with phosphate-bufferedsaline (PBS), scraped into 5 ml of ice-cold PBS and centrifuged at 200×gfor 5 minutes at 4° C. The pellet was resuspended in 2.5 ml ice-coldTris buffer (20mM Tris HC1, pH 7.4 at 23° C., 5 mM EDTA), handhomogenized in a Wheaton tissue grinder and the lysate centrifuged at200×g for 5 minutes at 4° C. to pellet large fragments. The supernatantwas then centrifuged at 40,000×g for 20 minutes at 4° C. The membraneswere washed once and resuspended in the homogenization buffer. Allpreparations were kept on ice and assays were run on the day on whichthe membranes were collected. Protein concentration was determined bythe method of Bradford (Anal. Biochem. 72: 248-54 (1976)) using bovineserum albumin as standard.

Radioligand Binding Studies

Binding assays were performed in triplicate in total volume of 250 μlcontaining buffer (50 mM Tris HCl, 10 mM MgSO₄, 1.5 mM EDTA, 150 mMNaCl, 0.1% ascorbate, 10 μM pargyline, pH 7.4 at 4° C.), [³H]SCH-23390(87 Ci/mmol; DuPont-NEN, Wilmington, Del.) and tested drugs. Incompetition binding experiments, 0.5-0.6 nM [³H]SCH-23390 was inhibitedby various concentrations of unlabeled drugs. Binding was initiated bythe addition of membrane preparation (10-20 μg protein) and carried outat 22° C. for 90 minutes. Specific binding was 95% of total binding at0.5 nM [³H]SCH-23390. For saturation experiments, membranes wereincubated with [³H]SCH-23390 over the concentration range of 0.01-6.5nM. Incubations were allowed to proceed for 150 minutes at 22° C. toensure that equilibrium was achieved at the lowest concentrations ofradioligand. Nonspecific binding was defined in the presence of 100 M(+) butaclamol. The reaction was terminated by rapid filtration throughWhatman GF/B glass fiber filters (presoaked with 0.5% polyethyleneamine,pH 7.4), using a Brandel 48R cell harvester (Brandel; Gaithersburg,Md.). Filters were washed for 5 seconds with iced buffer to reducenonspecific binding. Dried filters were transferred to scintillationvials and radioactivity was determined by liquid scintillation counting(Beckman LS 1701; Beckman Instruments, Fullerton, Calif.). Ready Safe(Beckman) was used as the scintillant and the counting efficiency was50%. Analysis of saturation and competition data were performed bycomputer-assisted nonlinear regression (DeLean et al., 1978; programsAccucomp and Accufit; Lundon Software, Chagrin Falls, Ohio). IC₅₀ valueswere converted to K_(i) values by the Cheng-Prusoff equation (Cheng andPrusoff, 1973).

Experimental Results Isolation of a Genomic Clone Encoding a DopamineDReceptor.

We have screened human genomic spleen and human genomic placentallibraries with the 1.6 kb Xba-1-Bam-H1 restriction fragment derived fromthe gene for the 5-HT1A receptor. A total of 59 clones were isolated andwere characterized by restriction endonuclease mapping. One clone(designated GL-30) was isolated as an approximately 4.0 kb EcoRI-Bgl-IIfragment was subcloned into pUC-18 and subject to sequence analysis.

Predicted Structure of the Receptor Encoded bv GL-30

DNA sequence information obtained from GL-30 is shown in FIGS. 1A-1E(SEQ ID NO: 1). An open reading frame encoding a protein of 477 aminoacids in length, having a relative molecular mass (M_(r)) ofapproximately 53 kD. A comparison of this protein sequence withpreviously characterized neurotransmitter receptors indicates that cloneGL-30 is a new member of a family of molecules which span the lipidbilayer seven times and couple to guanine nucleotide regulatory proteins(the G protein-coupled receptor family). A variety of structuralfeatures which are invariant in the G-protein coupled receptor family,including the aspartic acid residues of transmembrane regions II andIII, the DRY sequence at the end of transmembrane region III, and theconserved proline residues of transmembrane regions IV, V, VI and VIIwere present in clone GL-30. Both the amino terminus and theextracellular loop 2 (located between transmembrane domains IV and V) ofGL-30, contain consensus sites for N-linked glycosylation. In addition,this extracellular loop contains 45 amino acids (as compared to 31 aminoacids in the comparable region of the dopamine D₁ receptor) andrepresents the longest extracellular loop 2 of all the known G-proteincoupled receptors. While the carboxy-terminal tails of the dopamine D₁receptor and GL-30 are approximately the. same size, their amino acidsequences are only 41% identical. When compared to all the known Gprotein-coupled receptors, the greatest homology was found to be withthe dopamine D₁ receptor. While the overall homology between GL-30 andthe human dopamine D₁ receptor was 62%, the homology within the sevenmembrane spanning domains was 83% (FIGS. 2A-2E (SEQ ID NO: 2-4).

Discussion

Applicants have cloned and characterized a DNA molecule encoding a newdopamine D₁ receptor by low stringency hybridization to the serotonin5-HT_(1A) receptor. Although the amino acid sequence homology of cloneGL-30 to the 5-HT_(1A) receptor was relatively low (47% transmembraneregion identity), comparison of this sequence to previously cloneddopamine receptors showed that the closest relationship was to the humandopamine D₁ receptor (83% identity in the transmembrane domains). Incontrast, the transmembrane homology to either the dopamine D₂ ordopamine D₃ receptors was only 53% and 48%, respectively.

Clone GL-30 was expressed in Cos-7 cells in order to characterize thepharmacological binding properties of the expressed receptor protein.[³H]SCH-23390, a highly selective D₁ antagonist in the rat (Billard, W.et al. (1984) Life Sci. 35: 1885-93), non-human primate (Madras, B. K.et al. (1988) J. Neurochem. 51: 934-43) and human brain (DeKeyser, J. etal. (1988) Brain Res. 443: 77-84; Raisman, R. et al. (1985) Eur. J.Pharmacol. 113: 467-68) binds to this receptor with an apparentdissociation constant (K_(d)) of 0.65 nM, in good agreement with valuesreported for mammalian brain homogenates (Billard et al. (1984) supra;DeKeyser et al. (1988) supra; Raisman et al. (1985) supra; Reader, T. A.et al. (1989) Naunyn-Schmiedeberg's Arch. Pharmacol. 340: 617-25). Thisdissociation constant is nearly identical to that previously reportedfor the cloned D₁ receptor expressed in Cos-7 cells, K_(d)=0.3-06 nM,(Dearry et al. (1990) supra; Sunahara et al. (1990) supra; Zhou et al.(1990) supra).

Pharmacological characterization of the GL-30 clone showed binding of[³H]SCH-23390 to a site which clearly exhibited a D₁-like pharmacology(Table 1). The rank order of potencies of dopaminergic antagonists indisplacing the binding shows that the most potent compounds are thosepreviously identified as having the highest affinity for the D₁ site(e.g. SCH-23390, cis-flupenthixol and (+) butaclamol). Among other drugsclassified as dopamine receptor antagonists, bulbocapnine, haloperidoland clozapine yielded K_(i) values comparable to those reported for theD₁ receptor in native rat and human brain tissues, and for Cos-7 cellstransiently transfected with the previously cloned D₁ gene. The largestdifference found between the affinities of antagonists for this newlycloned receptor and those reported for the previously cloned D₁ receptorwas for (+) butaclamol which was 6-18 fold less potent at the dopamineDin receptor. Antagonist competition curves were of uniformly steepslope (n_(H)≈1.0) suggesting the presence of a single D₁ dopaminereceptor. The low affinity of (−) sulpiride and quinpirole to displace[³H]SCH-23390 binding is congruent with the D₂ selectivity of suchdrugs. The biogenic amine neurotransmitters serotonin and norepinephrinewere inactive in inhibiting the binding of the antagonist radioligand.

In contrast to the data on antagonist binding, the rank order ofpotencies and apparent dissociation constants obtained for dopaminergicagonists did not display a high degree of correlation with those foundin native brain tissues, in peripheral preparations, or in thepreviously characterized D₁ receptor clone. Dopamine displaced[³H]SCH-23390 binding with ≈10-20 fold higher affinity (K_(i)=159 nM)than that reported for D₁ receptors in either the brain or in theperiphery under the assay condition used. The competition curve fordopamine in these experiments had a relatively shallow slope, indicatingthe existence of both high and low affinity binding components. Theassay conditions were chosen to match those used in assays of thepreviously cloned D₁ receptor, and are expected to promote the lowaffinity configuration of the receptor. Although this dopamine D_(1B)receptor has pharmacological and functional properties similar to D₁receptors previously characterized in the brain and the periphery, itsagonist profile makes it a unique receptor.

Cos-7 cells transfected with clone GL-30 exhibited dopamine stimulatedcAMP production at a level 13 fold above the basal rate. This effect ofdopamine was blocked by the D₁ selective antagonist SCH-23390. The D₁selective partial agonist SKF-38393 stimulated cAMP accumulation to alesser extent than dopamine itself, consistent with its role as apartial agonist (Andersen et al. (1987) supra). The previously cloned D₁receptor was also shown to be coupled to stimulation of adenylatecyclase activity. The observation that the two different D₁ receptorgenes encode proteins which functionally couple to the same secondmessenger pathway reinforces the close relationship shown in their aminoacid sequences and pharmacological binding profiles. The existence oftwo separate genes with similar pharmacology and second messengercoupling suggests that their physiological roles may differ in someother aspect, such as tissue or cell-type distribution, synapticlocalization (postsynaptic v. presynaptic autoreceptor), ordevelopmental regulation.

Using gene specific primers for PCR amplification of RNA, thedistribution of messenger RNA encoding the dopamine D_(1B) receptor wasexamined. The dopamine D_(1B) receptor was found to be widelydistributed in a variety of higher brain centers, including brainstem,choroid plexus and hippocampus, suggesting a diverse role in regulatingbrain functions.

Clone GL-30 is an example of a G protein-coupled receptor whose entirecoding region is contained within a single exon, similar to the dopamineD₁ receptor (Dearry et al. (1990) supra; Monsma et al. (1990) supra;Sunahara et al. (1990) supra; Zhou et al. (1990) supra) and many othermembers of this superfamily. In contrast, the coding regions of thedopamine D₂ and D₃ receptors are interrupted by several introns (Bunzowet al., 1988; Sokoloff et al., 1990). Other subfamilies of Gprotein-coupled receptors (e.g. α₁ or α₂ adrenergic receptors), whichconsist of closely related subtypes, also share an intron-containing(α₁) or intronless nature (α₂) (Regan and Cotecchia, in press). Basedupon this similarity in intron-exon organization, as well as the closeamino acid homology to the previously cloned D₁ receptor,pharmacological binding properties, and second messenger coupling, cloneGL-30 can best be characterized as a dopamine D₁ receptor.

4 1771 base pairs nucleic acid single linear cDNA NO NO CDS 140..1573 1GCAGCTCATG GTGACCCCCC TCTGGGCTCG AGGGTCCCTT GGCTGAGGGG GCGCATCCTC 60GGGGTGCCGA TGGGGCTGCC TGGGGGTCGC AGGGCTGAAG TTGGGACCGC GCACAGACCG 120CCCCTGCAGT CCAGCCCAA ATG CTG CCG CCA GGC AGC AAC GGC ACC GCG TAC 172 MetLeu Pro Pro Gly Ser Asn Gly Thr Ala Tyr 1 5 10 CCG GGG CAG TTC GCT CTATAC CAG CAG CTG GCG CAG GGG AAC GCC GTG 220 Pro Gly Gln Phe Ala Leu TyrGln Gln Leu Ala Gln Gly Asn Ala Val 15 20 25 GGG GGC TCG GCG GGG GCA CCGCCA CTG GGG CCC TCA CAG GTG GTC ACC 268 Gly Gly Ser Ala Gly Ala Pro ProLeu Gly Pro Ser Gln Val Val Thr 30 35 40 GCC TGC CTG CTG ACC CTA CTC ATCATC TGG ACC CTG CTG GGC AAC GTG 316 Ala Cys Leu Leu Thr Leu Leu Ile IleTrp Thr Leu Leu Gly Asn Val 45 50 55 CTG GTG TGC GCA GCC ATC GTG CGG AGCCGC CAC CTG CGC GCC AAC ATG 364 Leu Val Cys Ala Ala Ile Val Arg Ser ArgHis Leu Arg Ala Asn Met 60 65 70 75 ACC AAC GTC TTC ATC GTG TCT CTG GCCGTG TCA GAC CTT TTC GTG GCG 412 Thr Asn Val Phe Ile Val Ser Leu Ala ValSer Asp Leu Phe Val Ala 80 85 90 CTG CTG GTC ATG CCC TGG AAG GCA GTC GCCGAG GTG GCC GGT TAC TGG 460 Leu Leu Val Met Pro Trp Lys Ala Val Ala GluVal Ala Gly Tyr Trp 95 100 105 CCC TTT GGA GCG TTC TGC GAC GTC TGG GTGGCC TTC GAC ATC ATG TGC 508 Pro Phe Gly Ala Phe Cys Asp Val Trp Val AlaPhe Asp Ile Met Cys 110 115 120 TCC ACT GCC TCC ATC CTG AAC CTG TGC GTCATC AGC GTG GAC CGC TAC 556 Ser Thr Ala Ser Ile Leu Asn Leu Cys Val IleSer Val Asp Arg Tyr 125 130 135 TGG GCC ATC TCC AGG CCC TTC CGC TAC AAGCGC AAG ATG ACT CAG CGC 604 Trp Ala Ile Ser Arg Pro Phe Arg Tyr Lys ArgLys Met Thr Gln Arg 140 145 150 155 ATG GCC TTG GTC ATG GTC GGC CTG GCATGG ACC TTG TCC ATC CTC ATC 652 Met Ala Leu Val Met Val Gly Leu Ala TrpThr Leu Ser Ile Leu Ile 160 165 170 TCC TTC ATT CCG GTC CAG CTC AAC TGGCAC AGG GAC CAG GCG GCC TCT 700 Ser Phe Ile Pro Val Gln Leu Asn Trp HisArg Asp Gln Ala Ala Ser 175 180 185 TGG GGC GGG CTG GAC CTG CCA AAC AACCTG GCC AAC TGG ACG CCC TGG 748 Trp Gly Gly Leu Asp Leu Pro Asn Asn LeuAla Asn Trp Thr Pro Trp 190 195 200 GAG GAG GAC TTT TGG GAG CCC GAC GTGAAT GCA GAG AAC TGT GAC TCC 796 Glu Glu Asp Phe Trp Glu Pro Asp Val AsnAla Glu Asn Cys Asp Ser 205 210 215 AGC CTG AAT CGA ACC TAC GCC ATC TCTTCC TCG CTC ATC AGC TTC TAC 844 Ser Leu Asn Arg Thr Tyr Ala Ile Ser SerSer Leu Ile Ser Phe Tyr 220 225 230 235 ATC CCC GTT GCC ATC ATG ATC GTGACC TAC ACG CGC ATC TAC CGC ATC 892 Ile Pro Val Ala Ile Met Ile Val ThrTyr Thr Arg Ile Tyr Arg Ile 240 245 250 GCC CAG GTG CAG ATC CGC AGG ATTTCC TCC CTG GAG AGG GCC GCA GAG 940 Ala Gln Val Gln Ile Arg Arg Ile SerSer Leu Glu Arg Ala Ala Glu 255 260 265 CAC GCG CAG AGC TGC CGG AGC AGCGCA GCC TGC GCG CCC GAC ACC AGC 988 His Ala Gln Ser Cys Arg Ser Ser AlaAla Cys Ala Pro Asp Thr Ser 270 275 280 CTG CGC GCT TCC ATC AAG AAG GAGACC AAG GTT CTC AAG ACC CTG TCG 1036 Leu Arg Ala Ser Ile Lys Lys Glu ThrLys Val Leu Lys Thr Leu Ser 285 290 295 GTG ATC ATG GGG GTC TTC GTG TGTTGC TGG CTG CCC TTC TTC ATC CTT 1084 Val Ile Met Gly Val Phe Val Cys CysTrp Leu Pro Phe Phe Ile Leu 300 305 310 315 AAC TGC ATG GTC CCT TTC TGCAGT GGA CAC CCT GAA GGC CCT CCG GCC 1132 Asn Cys Met Val Pro Phe Cys SerGly His Pro Glu Gly Pro Pro Ala 320 325 330 GGC TTC CCC TGC GTC AGT GAGACC ACC TTC GAC GTC TTC GTC TGG TTC 1180 Gly Phe Pro Cys Val Ser Glu ThrThr Phe Asp Val Phe Val Trp Phe 335 340 345 GGC TGG GCT AAC TCC TCA CTCAAC CCC GTC ATC TAT GCC TTC AAC GCC 1228 Gly Trp Ala Asn Ser Ser Leu AsnPro Val Ile Tyr Ala Phe Asn Ala 350 355 360 GAC TTT CAG AAG GTG TTT GCCCAG CTG CTG GGG TGC AGC CAC TTC TGC 1276 Asp Phe Gln Lys Val Phe Ala GlnLeu Leu Gly Cys Ser His Phe Cys 365 370 375 TCC CGC ACG CCG GTG GAG ACGGTG AAC ATC AGC AAT GAG CTC ATC TCC 1324 Ser Arg Thr Pro Val Glu Thr ValAsn Ile Ser Asn Glu Leu Ile Ser 380 385 390 395 TAC AAC CAA GAC ATC GTCTTC CAC AAG GAA ATC GCA GCT GCC TAC ATC 1372 Tyr Asn Gln Asp Ile Val PheHis Lys Glu Ile Ala Ala Ala Tyr Ile 400 405 410 CAC ATG ATG CCC AAC GCCGTT ACC CCC GGC AAC CGG GAG GTG GAC AAC 1420 His Met Met Pro Asn Ala ValThr Pro Gly Asn Arg Glu Val Asp Asn 415 420 425 GAC GAG GAG GAG GGT CCTTTC GAT CGC ATG TTC CAG ATC TAT CAG ACG 1468 Asp Glu Glu Glu Gly Pro PheAsp Arg Met Phe Gln Ile Tyr Gln Thr 430 435 440 TCC CCA GAT GGT GAC CCTGTT GCT GAG TCT GTC TGG GAG CTG GAC TGC 1516 Ser Pro Asp Gly Asp Pro ValAla Glu Ser Val Trp Glu Leu Asp Cys 445 450 455 GAG GGG GAG ATT TCT TTAGAC AAA ATA ACA CCT TTC ACC CCG AAT GGA 1564 Glu Gly Glu Ile Ser Leu AspLys Ile Thr Pro Phe Thr Pro Asn Gly 460 465 470 475 TTC CAT TAAACTGCATTAA GAACCCTCAT GGATCTGCAT AACCGCACAG 1613 Phe His * ACACTGACAAGCACGCACAC ACACGCAAAT ACATGCCTTT CAGTGCTGCT CCTTATCATG 1673 TGTTCTGTGTAGTAGCTCGT GTGCTAGAAC TCACCATGAT GTCAGTCGAG ATGCAGATCA 1733 GTGCATACTCAGTCAAGTAT CAGCTACAGA GATGACAC 1771 477 amino acids amino acid linearprotein 2 Met Leu Pro Pro Gly Ser Asn Gly Thr Ala Tyr Pro Gly Gln PheAla 1 5 10 15 Leu Tyr Gln Gln Leu Ala Gln Gly Asn Ala Val Gly Gly SerAla Gly 20 25 30 Ala Pro Pro Leu Gly Pro Ser Gln Val Val Thr Ala Cys LeuLeu Thr 35 40 45 Leu Leu Ile Ile Trp Thr Leu Leu Gly Asn Val Leu Val CysAla Ala 50 55 60 Ile Val Arg Ser Arg His Leu Arg Ala Asn Met Thr Asn ValPhe Ile 65 70 75 80 Val Ser Leu Ala Val Ser Asp Leu Phe Val Ala Leu LeuVal Met Pro 85 90 95 Trp Lys Ala Val Ala Glu Val Ala Gly Tyr Trp Pro PheGly Ala Phe 100 105 110 Cys Asp Val Trp Val Ala Phe Asp Ile Met Cys SerThr Ala Ser Ile 115 120 125 Leu Asn Leu Cys Val Ile Ser Val Asp Arg TyrTrp Ala Ile Ser Arg 130 135 140 Pro Phe Arg Tyr Lys Arg Lys Met Thr GlnArg Met Ala Leu Val Met 145 150 155 160 Val Gly Leu Ala Trp Thr Leu SerIle Leu Ile Ser Phe Ile Pro Val 165 170 175 Gln Leu Asn Trp His Arg AspGln Ala Ala Ser Trp Gly Gly Leu Asp 180 185 190 Leu Pro Asn Asn Leu AlaAsn Trp Thr Pro Trp Glu Glu Asp Phe Trp 195 200 205 Glu Pro Asp Val AsnAla Glu Asn Cys Asp Ser Ser Leu Asn Arg Thr 210 215 220 Tyr Ala Ile SerSer Ser Leu Ile Ser Phe Tyr Ile Pro Val Ala Ile 225 230 235 240 Met IleVal Thr Tyr Thr Arg Ile Tyr Arg Ile Ala Gln Val Gln Ile 245 250 255 ArgArg Ile Ser Ser Leu Glu Arg Ala Ala Glu His Ala Gln Ser Cys 260 265 270Arg Ser Ser Ala Ala Cys Ala Pro Asp Thr Ser Leu Arg Ala Ser Ile 275 280285 Lys Lys Glu Thr Lys Val Leu Lys Thr Leu Ser Val Ile Met Gly Val 290295 300 Phe Val Cys Cys Trp Leu Pro Phe Phe Ile Leu Asn Cys Met Val Pro305 310 315 320 Phe Cys Ser Gly His Pro Glu Gly Pro Pro Ala Gly Phe ProCys Val 325 330 335 Ser Glu Thr Thr Phe Asp Val Phe Val Trp Phe Gly TrpAla Asn Ser 340 345 350 Ser Leu Asn Pro Val Ile Tyr Ala Phe Asn Ala AspPhe Gln Lys Val 355 360 365 Phe Ala Gln Leu Leu Gly Cys Ser His Phe CysSer Arg Thr Pro Val 370 375 380 Glu Thr Val Asn Ile Ser Asn Glu Leu IleSer Tyr Asn Gln Asp Ile 385 390 395 400 Val Phe His Lys Glu Ile Ala AlaAla Tyr Ile His Met Met Pro Asn 405 410 415 Ala Val Thr Pro Gly Asn ArgGlu Val Asp Asn Asp Glu Glu Glu Gly 420 425 430 Pro Phe Asp Arg Met PheGln Ile Tyr Gln Thr Ser Pro Asp Gly Asp 435 440 445 Pro Val Ala Glu SerVal Trp Glu Leu Asp Cys Glu Gly Glu Ile Ser 450 455 460 Leu Asp Lys IleThr Pro Phe Thr Pro Asn Gly Phe His 465 470 475 477 amino acids aminoacid single linear peptide 3 Met Leu Pro Pro Arg Ser Asn Gly Thr Ala TyrPro Gly Gln Leu Ala 1 5 10 15 Leu Tyr Gln Gln Leu Ala Gln Gly Asn AlaVal Gly Gly Ser Ala Gly 20 25 30 Ala Pro Pro Leu Gly Pro Val Gln Val ValThr Ala Cys Leu Leu Thr 35 40 45 Leu Leu Ile Ile Trp Thr Leu Leu Gly AsnVal Leu Met Ser Ala Ala 50 55 60 Ile Val Arg Thr Arg His Leu Arg Ala LysMet Thr Asn Val Phe Ile 65 70 75 80 Val Ser Leu Ala Val Ser Asp Leu PheVal Ala Leu Leu Val Met Pro 85 90 95 Trp Lys Ala Val Ala Glu Val Ala GlyTyr Trp Pro Phe Glu Ala Phe 100 105 110 Cys Asp Val Trp Val Ala Phe AspIle Met Cys Ser Thr Ala Ser Ile 115 120 125 Leu Asn Leu Cys Val Ser ValIle Ser Val Gly Arg Tyr Trp Ala Ile 130 135 140 Ser Arg Pro Phe Arg TyrGlu Arg Lys Met Thr Gln Arg Met Ala Leu 145 150 155 160 Val Met Val GlyPro Ala Trp Thr Leu Ser Ser Leu Ile Ser Phe Ile 165 170 175 Pro Val GlnLeu Asn Trp His Arg Asp Gln Ala Val Ser Gly Gly Leu 180 185 190 Asp LeuPro Asn Asn Leu Ala Asn Trp Thr Pro Trp Glu Glu Ala Val 195 200 205 TrpGlu Pro Asp Val Arg Ala Glu Asn Cys Asp Ser Ser Leu Asn Arg 210 215 220Thr Tyr Ala Ile Ser Ser Ser Leu Ile Asn Phe Tyr Ile Pro Met Ala 225 230235 240 Ile Met Ile Val Thr Tyr Thr Arg Ile Tyr Arg Ile Ala Gln Val Gln245 250 255 Ile Cys Arg Ile Ser Ser Leu Glu Arg Ala Ala Glu His Val GlnSer 260 265 270 Cys Arg Ser Ser Ala Gly Cys Thr Pro Asp Thr Ser Leu ArgPhe Ser 275 280 285 Ile Lys Lys Glu Thr Lys Val Leu Lys Pro Leu Ser ValIle Met Gly 290 295 300 Val Phe Val Cys Cys Trp Leu Pro Phe Phe Ile LeuAsn Cys Met Val 305 310 315 320 Pro Phe Arg Ser Gly His Pro Lys Gly ProPro Ala Gly Phe Pro Cys 325 330 335 Val Ser Glu Thr Thr Phe Asp Val PheIle Trp Phe Cys Trp Ala Asn 340 345 350 Ser Ser Leu Asn Pro Val Tyr AlaPhe Asn Ala Asp Phe Trp Lys Val 355 360 365 Phe Ala Gln Leu Leu Gly CysSer His Val Cys Ser Arg Thr Pro Val 370 375 380 Glu Thr Val Asn Ile SerAsn Glu Leu Ile Ser Tyr Asn Gln Asp Met 385 390 395 400 Val Phe His LysGlu Ile Ala Ala Ala Cys Ile His Met Met Pro Asn 405 410 415 Ala Val ProPro Gly Asp Gln Glu Val Asp Asn Asp Glu Glu Glu Glu 420 425 430 Ser ProPhe Asp Arg Met Ser Gln Ile Tyr Gln Thr Ser Pro Asp Gly 435 440 445 AspPro Val Ala Glu Ser Val Glu Leu Asp Cys Glu Gly Glu Ile Ser 450 455 460Leu Asp Lys Ile Thr Pro Phe Thr Pro Asn Gly Phe His 465 470 475 446amino acids amino acid single linear peptide D1 4 Met Arg Thr Leu AsnThr Ser Ala Met Asp Gly Thr Gly Leu Val Val 1 5 10 15 Glu Arg Asp PheSer Val Arg Ile Leu Thr Ala Cys Phe Leu Ser Leu 20 25 30 Leu Ile Leu SerThr Leu Leu Gly Asn Thr Leu Val Cys Ala Ala Val 35 40 45 Ile Arg Phe ArgHis Leu Arg Ser Lys Val Thr Asn Phe Phe Val Ile 50 55 60 Ser Leu Ala ValSer Asp Leu Leu Val Ala Val Leu Val Met Pro Trp 65 70 75 80 Lys Ala ValAla Glu Ile Ala Gly Phe Trp Pro Phe Gly Ser Phe Cys 85 90 95 Asn Ile TrpVal Ala Phe Asp Ile Met Cys Ser Thr Ala Ser Ile Leu 100 105 110 Asp LeuCys Val Ile Ser Val Asp Arg Tyr Trp Ala Ile Ser Ser Pro 115 120 125 PheArg Tyr Glu Arg Lys Met Thr Pro Lys Ala Ala Phe Ile Leu Ile 130 135 140Ser Val Ala Trp Thr Leu Ser Val Leu Ile Ser Phe Ile Pro Val Gln 145 150155 160 Leu Ser Trp His Lys Ala Lys Pro Thr Ser Pro Ser Asp Gly Asn Ala165 170 175 Thr Ser Leu Ala Glu Thr Ile Asp Asn Cys Asp Ser Ser Leu SerArg 180 185 190 Thr Tyr Ala Ile Ser Ser Ser Val Ile Ser Phe Tyr Ile ProVal Ala 195 200 205 Ile Met Ile Val Thr Tyr Thr Arg Ile Tyr Arg Ile AlaGln Lys Gln 210 215 220 Ile Arg Arg Ile Ala Ala Leu Glu Arg Ala Ala ValHis Ala Lys Asn 225 230 235 240 Cys Gln Thr Thr Thr Gly Asn Gly Lys ProVal Glu Cys Ser Gln Pro 245 250 255 Glu Ser Ser Phe Lys Met Ser Phe LysArg Glu Thr Lys Val Leu Lys 260 265 270 Thr Leu Ser Val Ile Met Gly ValPhe Val Cys Cys Trp Leu Pro Phe 275 280 285 Phe Ile Leu Asn Cys Ile LeuPro Phe Cys Gly Ser Gly Glu Thr Gln 290 295 300 Pro Phe Cys Ile Asp SerAsn Thr Phe Asp Val Phe Val Trp Phe Gly 305 310 315 320 Trp Ala Asn SerSer Leu Asn Pro Ile Ile Tyr Ala Phe Asn Ala Asp 325 330 335 Phe Arg LysAla Phe Ser Thr Leu Leu Gly Cys Tyr Arg Leu Cys Pro 340 345 350 Ala ThrAsn Asn Ala Ile Glu Thr Val Ser Ile Asn Asn Asn Gly Ala 355 360 365 AlaMet Phe Ser Ser His His Glu Pro Arg Gly Ser Ile Ser Lys Glu 370 375 380Cys Asn Leu Val Tyr Leu Ile Pro His Ala Val Gly Ser Ser Glu Asp 385 390395 400 Leu Lys Lys Glu Glu Ala Ala Gly Ile Ala Arg Pro Leu Glu Lys Leu405 410 415 Ser Pro Ala Leu Ser Val Ile Leu Asp Tyr Asp Thr Asp Val SerLeu 420 425 430 Glu Lys Ile Gln Pro Ile Thr Gln Asn Gly Gln His Pro Thr

What is claimed is:
 1. A purified dopamine D_(1B) receptor proteinhaving the amino acid sequence set forth in Seq. I.D. No.
 2. 2. A methodof preparing the isolated dopamine D_(1B) receptor protein of claim 1which comprises: a. inducing cells which comprise an expression plasmidto express the dopamine D_(1B) receptor protein, wherein the plasmidcomprises a recombinant vector comprising an isolated nucleic acidencoding the human dopamine D_(1B) receptor having the amino acidsequence set forth in Seq. I.D. No. 2 or an amino acid sequenceidentical to that encoded by a nucleic acid sequence contained inplasmid pdopD1-GL-30 (ATCC Accession No. 40839); b. recovering theD_(1B) receptor protein expressed by the cells; and c. purifying theD_(1B) receptor protein.
 3. A method of preparing a purified dopamineD_(1B) receptor protein which comprises: a. inserting a nucleic acidinto a suitable expression vector such that the dopamine D_(1B) receptorprotein may be expressed in a suitable host cell, wherein the nucleicacid encodes the human dopamine D_(1B) receptor having the amino acidsequence shown in Seq. I.D. No. 2 or an amino acid sequence identical tothat encoded by a nucleic acid sequence contained in plasmidpdopD1-GL-30 (ATCC Accession No. 40839); b. introducing the resultingvector into the suitable host cell so that the dopamine D_(1B) receptorprotein is expressed by the host cell; c. recovering the D_(1B) receptorprotein expressed by the resulting cell; and d. purifying the D_(1B)receptor protein.